Disregulation of Src family kinases (SFKs) plays an important role in tumor development, specifically metastasis. Current therapeutic development targets the enzymatic activity of these signaling molecules. However, this may not completely block the aberrant activities of these oncogenic proteins. A recent unbiased screen of kinase mutations in cancers revealed a large number of inactivating mutations indicating important non-catalytic functions of kinases in cancer biology. Accumulating evidence suggests that non-catalytic roles of protein kinases such as scaffolding, subcellular targeting or DNA binding, are essential and in some cases sufficient for function. The long term goal of our study is to gain better understanding of the non-catalytic functions of SFK members which will guide a better therapeutic approach to cancers involving SFK signaling pathways. In addition to catalytic activity, kinases can act as scaffolding molecules via protein-protein interactions through SH2 and SH3 domains. Accessibility of these domains can be regulated through conformational changes which are stabilized by ligand binding in the ATP-binding site. In this proposal, the availability of the SH2 and SH3 domains of SFKs for protein-protein interactions and accessibility for post- translational modifications in multiple ATP-binding site conformations will be determined using two sets of biochemical assays. First, fluorescence polarization, limited proteolysis and pull-down assays will be used to probe the accessibility of the SH2 and SH3 domains to inter-molecular binding partners. Second, a series of enzymatic assays with regulatory kinases and phosphatases will investigate the susceptibility of inhibitor- bound SFKs to post-translational modifications. In order to study the effects of varying SH2 and SH3 domain accessibility to inter-molecular interactions in a complex biological system, a chemical genetic approach using orthogonal kinase ligands capable of stabilizing specific ATP-binding site conformations of drug-sensitized Src family kinases. Finally, the effects of stabilizing distinct SFK ATP-binding site conformations on Src/FAK complex formation, phosphorylation of complex substrates, and rescue of cell motility will be investigated in SFK-knockout mouse embryonic fibroblasts expressing drug-sensitized Src or Fyn gatekeeper mutants. The ability to modulate protein-protein interactions outside of the ATP binding site with ATP-competitive inhibitors will allow greater control of kinase function and has the potential to selectively inhibt specific signaling pathways. This study should guide the future therapeutic design of SFK inhibitors in aberrant signaling networks by developing inhibitors which selectively or cooperatively inactivate catalytic and non-catalytic functions. Furthermore, the technology developed in this proposal will be widely applicable to other kinase families, providing a powerful tool in understanding kinase function in oncogenic systems.